We propose to study the structure and the role of microtubules in mammalian cells using a combined biochemical and genetic approach. Our basic strategy will be to isolate mutants in tubulin or in microtubule associated proteins (MAPs) by looking for resistance of Chinese hamster ovary (CHO) cells to drugs which are known to interact with microtubules and prevent their polymerization or inhibit their function. These mutants will first be characterized for their biochemical and morphological alterations, and then be used to look at a variety of cellular functions in which microtubules have been implicated. The goal of these studies will be to place the involvement of microtubules in these cellular functions on a firmer basis and to help define the mechanism of microtubule action. Identification of the biochemical lesion in the mutant cells will be made using two-dimensional gel electrophoresis of whole cell extracts and of crude microtubule preparations. Mutants with a demonstrated alteration in tubulin or MAPs will be further characterized for their dominance, cross-resistance to anti-mitotic drugs, and temperature sensitivity (ts) of growth, and for gross alterations in microtubule structure using electron microscopy and indirect immunofluorescence with atiserum to tubulin. For those mutants which are ts for growth, temperature resistant revertants will be isolated and tested for co-reversion of their drug resistance and alteration in tubulin or MAPs. These revertants will be used to prove that the alteration in tubulin or MAPs is responsible for the drug-resistance and temperature sensitivity of the cell. Attempts will be made to isolate second site revertants as a way of identifying proteins that interact to form microtubules. These well characterized mutants will be used to explore the role of microtubules in a variety of cellular processes such as mitosis, saltatory motion, and hormonal stimulation, and to examine possible interactions between microtubules and intermediate filaments or calmodulin.